The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores.

Abstract

P2Y(2) receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemoattractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide. UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2 receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126, supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca(2+)-ATPases in mammalian cells, the effect of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils.

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The priming effect of extracellular UTP on human neutrophils:
Role of calcium released from thapsigargin-sensitive intracellular stores
Florin Tuluc1, Ovidiu Bredetean1, Eugen Brailoiu2, John Meshki1, Analia Garcia1, Nae J. Dun2
& Satya P. Kunapuli1
1Department of Physiology, Temple University Medical School, Philadelphia, Pennsylvania, USA;
2Department of Pharmacology, Temple University Medical School, Philadelphia, Pennsylvania, USA
Received 22 February 2005; accepted in revised form 3 June 2005
Key words: calcium, mitogen-activated protein kinases, neutrophil, P2Y receptors, reactive oxygen species
Abstract
P2Y2receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as
intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear
leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide
production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular
nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemo-
attractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS
production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to
chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide.
UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2
receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126,
supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence
of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca2+-ATPases in mammalian cells, the effect
of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from
thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils.
Introduction
Although ATP is abundantly present in living cells, it is
mainly confined to the intracellular environment where it
commonly reaches concentrations in low millimolar range
[1]. ATP is found at even higher concentrations (approx-
imately 150 mM) in chromaffin cells of the adrenal
medulla [2]. There is strong evidence indicating that
endothelial cells, activated platelets, sympathetic nerve
endings as well as damaged cells can release significant
amounts of ATP in the extracellular medium under a
variety of pathological conditions such as atherosclerosis,
hypertension, ischemia, and inflammation [1, 3]. UTP,
although not as abundant as ATP, has been reported to be
released from platelets, leukocytes, primary airway epithe-
lial cells, rat astrocytes and several cell lines [4, 5]. Most
mammalian cell types, including leukocytes, express on the
plasma membrane receptors for extracellular nucleotides as
well as ectonucleotidases, enzymes which are able to
quickly hydrolyze nucleotides [6]. It has been previously
shown that ATP causes an enhancement of N-formyl-
methionyl-leucyl-phenylalanine (fMLP)-stimulated super-
oxide anion (O2
polymorphonuclear neutrophils at concentrations similar to
those attained extracellularly at sites of platelet thrombus
formation (0.1 to 20 mM). However, ATP by itself was
unable to cause superoxide anion generation in isolated
neutrophils [8]. Priming of the neutrophil respiratory burst
has been implicated in the pathogenesis of multi-system
organ failure after sepsis and trauma and appears to be
mediated by multiple signaling pathways, depending on the
particular priming agent and on the agonist [9]. MAP
kinases have been shown to participate in the priming
effect of TNF-a and granulocyte-macrophage colony-
stimulating factor (GM-CSF) but not in the case of
platelet-activating factor (PAF) [9].
We have previously shown that the main nucleotide
receptor mediating nucleotide-induced responses in neu-
trophils is the P2Y2receptor, which is equally responsive
to ATP and UTP [10]. In the present study we performed a
pharmacological characterization of the priming effect of
nucleotides on ROS production induced by fMLP or IL-8.
We also explored the effect of extracellular nucleotides on
j) generation [7, 8] when added to human
Correspondence to: Dr Satya P. Kunapuli PhD, Department of Physiol-
ogy, Temple University Medical School, 3420 N. Broad Street, OMS 224,
Philadelphia, PA 19140, USA. Tel: +1-215-707-4615; Fax: +1-215-707-
4003; E-mail: spk@temple.edu
Purinergic Signalling (2005) 1: 359–368
DOI: 10.1007/s11302-005-0039-8
#Springer 2005

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several intracellular signaling pathways involved in the
potentiation effect of nucleotides. We found that although
the phosphorylation of p44/42 (Erk) and p38 MAP kinases
is enhanced by extracellular nucleotides, this event does
not participate in priming. In addition, we investigated the
role played by free cytosolic calcium in the priming effect of
nucleotides. In isolated neutrophils, nucleotides cause
increase of cytosolic free calcium by release from intracel-
lular stores and through influx [11]. In calcium-depleted
neutrophils fMLP-induced ROS production was signifi-
cantly inhibited but the potentiating effect of nucleotides
was still present, although not as robust as in cells main-
tained in medium containing physiological concentrations
of calcium.
We showed for the first time that thapsigargin, a widely
usedinhibitoroftheubiquitoussarco-endoplasmicreticulum
Ca2+-ATPases in mammalian cells, is able to selectively
block the potentiating effect of nucleotides on fMLP-
induced ROS production. Our data support the view that
the release of calcium from thapsigargin-sensitive intracel-
lular stores is essential for nucleotide-induced priming.
Materials and methods
Reagents.
2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole),
U0126 (1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenyl-
mercapto) butadiene), brefeldin A, thapsigargin, and
bovine serum albumin (BSA; fraction V) were obtained
from Sigma (St. Louis, Missouri, USA). Dextran T500,
Ficoll-Paque were from Amersham Biosciences (Piscat-
All nucleotides, SB203580 (4-(4-Fluorophenyl)-
away, New Jersey, USA). CDP-Star with Nitro-Block II\
from Tropix (Bedford, Massachusetts, USA) was used for
chemiluminescent detection in Western blotting experi-
ments. Fura-2 AM was from Molecular Probes (Eugene,
Oregon, USA) and IL-8 was from Peprotech (Rocky Hill,
New Jersey, USA). Polyclonal anti phospho-p44/42 MAP
kinase (Thr202/Tyr204), anti-p44/442 MAP kinase, anti
phospho-p38 MAP kinase (Thr180/Tyr182) antibodies, and
anti p38 MAP kinase were from Cell Signaling Technology
(Beverly, Massachusetts, USA).
Neutrophil isolation was performed as previously described
[12]. Briefly, venous blood was collected upon written
consent from healthy subjects in polypropylene tubes
containing ACD anticoagulant (1.5% citric acid, 2.5%
sodium citrate, 2% dextrose). Blood was mixed with an
equal volume of 3% dextran T500 in saline. Erythrocytes
were allowed to sediment for 20 min then leukocyte rich
plasma was subjected to centrifugation on Ficoll-Paque at
400?g for 45 min. The pellet was collected and the
contaminating erythrocytes were removed by hypotonic
lysis. Isolated neutrophils were resuspended in Hank’s
balanced salt solution (HBSS) containing 0.2% BSA.
Neutrophils were counted using a Reichert-Jung hema-
cytometer (Hausser Scientific, Horsham, Pennsylvania,
USA). Cell viability was checked by the Trypan blue
exclusionmethod andwas routinely foundgreaterthan96%.
Measurement of cytoplasmic free Ca2+concentration.
Isolated neutrophils were incubated for 45 min at room
temperature in HBSS containing bovine serum albumin
Figure 1.
was measured as described under Materials and methods. (A) and (B) The effect of fMLP (10 nM) or IL-8 (10 nM) was measured when administered
alone (empty bars) or simultaneously with 10 mM UTP (closed bars). (C) and (D) The effect of fMLP or IL-8 was measured when added at different
intervals after the addition of UTP. Averages T SEM of peak luminescence signal measured in 4Y6 experiments are shown. The luminescence peak
measured upon the addition of a final concentration of 200 mM of H2O2to medium containing isoluminol (5 mM) and horseradish peroxidase (1 U/ml)
corresponds to 100 U on the Y-axes. (*P G 0.05).
UTP potentiates fMLP- or IL-8-induced ROS production in a time-dependent manner. Human neutrophils were isolated and ROS production
360
F. Tuluc et al.

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0.2% and 1 mM Fura-2 AM. The cells were washed twice
and then resuspended at a concentration of 3 ? 106/ml in
HBSS containing BSA 0.2% and cytoplasmic free Ca2+
concentration was measured as previously described [12].
Briefly, aliquots of 0.5 ml were placed in disposable
methacrylate cuvettes under stirring conditions at 37 -C.
Intracellular calcium concentrations were recorded during
agonist stimulation using excitation wavelengths of 340
and 380 nm and the emission was monitored at 510 nm
using an AB2 luminescence spectrophotometer (Spec-
tronics Instruments, Rochester, New York, USA). Cyto-
plasmic concentrations of calcium were calculated
according to Tsien’s ratiometric method [13]. Averages of
peak intracellular calcium concentrations were compared.
Measurement of ROS production.
was measured as described elsewhere [14]. Briefly,
horseradish peroxidase (1 U/ml) and isoluminol (5 mM)
were added to neutrophil suspensions (2 ? 106/ml) in a
glass cuvette under stirring conditions, at 37 -C in a lumi-
aggregometer model 560-CA (Chronolog Corp., Haver-
town, Pennsylvania, USA). The cells were allowed to
equilibrate for several minutes and reagents were added to
the cell suspension while recording the luminescence
signal on a chart recorder. For standardization purposes,
hydrogen peroxide was used to assess the sensitivity of the
peroxidase-isoluminol system and for normalization of data.
The luminescence peak measured upon the addition of a
final concentration of 200 mM of H2O2 to medium
containing isoluminol (5 mM) and horseradish peroxidase
(1 U/ml) was considered equal to 100 U. Averages of peak
luminescence signals were compared.
The release of ROS
Western blotting.
were incubated with UTP (10 mM) or solvent for 1 min at
37 -C then cells were stimulated with 10 nM fMLP or
IL-8 for 2 min. The stimulation was terminated by adding
to each sample an equal volume of 2? lysis buffer con-
taining a mixture of protease and phosphatase inhibitors.
The final concentrations of reagents in the lysis medium
were: 25 mM TrisYHCl, 150 mM NaCl, 5 mM EDTA, 1%
Triton X-100, 1 mM PMSF, 10 mg/ml leupeptin, 10 mg/ml
aprotinin, 1 mM sodium vanadate, 2 mM sodium pyro-
phosphate, 10 mM b-glycerol phosphate. The lysis was
performed on ice for 20 min followed by centrifugation of
the samples at 13,000 rpm for 5 min. A volume of 200
ml of the cytosolic fraction (the supernatant) was collected
and mixed with 100 ml of sample buffer containing: 6%
sodium dodecyl sulphate, 30% glycerol, 3 mM EDTA,
0.03% phenol red. Proteins were separated by SDS-
polyacrylamide gel electrophoresis, transferred to Immun-
Blot\PVDF membrane (Bio-Rad, Hercules, California,
USA) and incubated for 30 min in Tris buffered saline
(TTBS; 20 mM Tris, 150 mM NaCl, 0.05% Tween 20; pH
7.5) containing 2% BSA (blocking buffer). Membranes
were incubated overnight at 4 -C in the presence of the
indicated primary antibody, which was diluted 1:1,000 in
blocking buffer. Membranes were washed and incubated
with appropriate secondary antibodies conjugated with
Isolated humanneutrophils(6?106/ml)
alkaline phosphatase for 30 min. Membranes were exten-
sively washed and CDP-Star with Nitro-Block II\(Tropix,
Bedford, Massachusetts, USA) was used for chemilumi-
nescent detection of the reactive proteins on a Fujifilm
LAS-1000 CCD system.
Data analysis.
one way analysis of variance (ANOVA). A P value G 0.05
was considered significant.
Statistical comparisons were made using
Results
Nucleotides potentiate ROS production induced by fMLP
or IL-8 in a time- and dose- dependent manner
We performed a pharmacological characterization of the
potentiating effect of extracellular nucleotides on the
production of reactive oxygen species caused by two
well-known chemoattractants, fMLP and IL-8. We tested
ATP and UTP, two nucleotides that have been shown to be
released in the extracellular medium under physiological or
pathological conditions. When added alone to neutrophil
suspensions, ATP and UTP did not cause measurable ROS
production. However, both nucleotides were able to
enhance the production of ROS induced by either IL-
Figure 2.
production in a dose-dependent manner. Human neutrophils were isolated
and ROS production was measured as described under Materials and
methods. The effect of 10 nM fMLP (A) or 10 nM IL-8 (B) was measured
when administered alone or 1 min after the addition of increasing
concentrations of UTP (circles) or ATP (squares). Averages T SEM of
peak luminescence signal measured in 3Y5 experiments are shown. The
luminescence peak measured upon the addition of a final concentration of
200 mM of H2O2to medium containing isoluminol (5 mM) and horseradish
peroxidase (1 U/ml) corresponds to 100 U on the Y-axes. (*P G 0.05 when
compared to the values measured in the absence of nucleotides.)
UTP and ATP potentiate fMLP- or IL-8-induced ROS
The priming effect of nucleotides in human neutrophils
361

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8 (10 nM) or fMLP (10 nM). When a mixture of UTP and
chemoattractant was added to neutrophil suspensions, a
significant potentiation of the effect of chemoattractant was
measured (Figures 1A and B). The potentiation of ROS
production induced by IL-8 was higher (2.6-fold increase)
as compared to the effect of fMLP (1.7-fold). In order to
determine if the potentiation effect of nucleotides is time-
dependent, UTP (10 mM) was added to neutrophil
suspension at different intervals prior the addition of fMLP
(10 nM) or IL-8 (10 nM). The maximal potentiating effect
of UTP was detected when the addition of nucleotide was
performed at 1 min before the addition of the chemo-
attractant (Figures 1C and D).
The relationship between the concentration of nucleotide
and the priming effect was assessed by administering
different concentrations of nucleotide at a fixed interval (1
min) before the addition of chemoattractant (Figure 2). The
maximal effect of nucleotides was obtained at 10 mM,
which is consistent with the potency described for these
agonists at P2Y2 receptors expressed in 1321N1 cells,
which are devoid of endogenous P2 receptors [15].
Nucleotides potentiate the phosphorylation of MAP kinases
induced by fMLP and IL-8
MAP kinases have been reported to play a role in
mediating neutrophil functions. In the present study we
Figure 3.
solvent for 1 min at 37 -C then cells were stimulated for 2 min with fMLP or IL-8, as indicated. Cells were lysed and solubilized proteins were separated
by SDS-polyacrylamide gel electrophoresis. The detection of phosphorylated MAP kinases was performed as described under Materials and methods.
(C) Densitometric analysis of images obtained from three Western blotting experiments. Data were normalized for the maximum effect obtained in cells
stimulated with UTP and fMLP, which was considered equal to 100 arbitrary units (AU).
UTP induces MAP kinase phosphorylation. (A) and (B) Isolated human neutrophils (6 million/ml) were incubated with UTP (10 mM) or
Figure 4.
the potentiating effect of nucleotides on ROS production. Isolated
neutrophils were incubated with SB203580 (10 mM) or U0126 (3 mM)
for 15 min then ROS production was measured as described under
Materials and methods. The effect of fMLP (10 nM) or IL-8 (10 nM) was
determined when administered 1 min after the addition of UTP (10 mM;
closed bars) or solvent (open bars). Averages T SEM of peak lumines-
cence signal measured in 4 Y5 experiments are shown (*P G 0.05).
Inhibition of p38 or p44/42 MAP kinase has minor effect on
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F. Tuluc et al.

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investigated the role of p44/42 and p38 MAP kinases in
nucleotide-induced potentiation of ROS production. Both
p44/42 and p38 MAP kinases have been shown to be
activated by upstream kinases, which are able to phosphor-
ylate specific residues. The p44/42 MAP kinase is activated
by dual phosphorylation at Thr202 and Tyr204 [16], while
p38 MAP kinase activation requires dual phosphorylation
at Thr180 and Tyr182 [17]. We determined the phosphor-
ylation of p44/42 and p38 MAP kinases by Western
blotting, using phospho-specific antibodies.
UTP was able to weakly phosphorylate p44/42 MAP
kinase and had a more prominent effect on the phosphor-
ylation of p38 MAPK (Figure 3). We have previously
shown that ATP had a similar effect on the phosphoryla-
tion of MAP kinases [10] consistent with the view that
ATP and UTP act on a common receptor on human
neutrophils. When added to neutrophils 60 s prior to the
Table 1.
ROS production in human neutrophils.
The effect of MAP kinase inhibitors on fMLP- or IL-8-induced
ControlSB203580 (10 mM)U0126 (3 mM)
Solvent + fMLP
UTP + fMLP
Fold increase
25.3 T 2.3
95.0 T 14.0
3.8
8.5 T 2.5
54.6 T 19.7
6.4
9.5 T 1.5
80.0 T 18.4
8.4
Solvent + IL-8
UTP + IL-8
Fold increase
18.0 T 5.6
79.0 T 7.0
4.4
6.4 T 2.8
42.7 T 9.3
6.7
5.9 T 4.3
42.4 T 17.4
7.2
ROS production was measured in isolated human neutrophils as described
under Materials and methods. Cells were incubated with UTP (10 mM) or
solvent then fMLP (10 nM) or IL-8 (10 nM) were added to the medium.
Averages T SEM of peak luminescence signal are presented. The
luminescence peak measured upon the addition of a final concentration
of 200 mM of H2O2to medium containing isoluminol (5 mM) and horse-
radish peroxidase (1 U/ml) corresponds to 100 U.
Figure 5.
neutrophils were isolated and intracellular calcium increase was measured as described under Materials and methods. The effect of fMLP (10 nM) or IL-
8 (10 nM) was measured when administered 2 min after the addition of different concentrations of UTP (circles) or ATP (squares). Averages T SEM of
peak intracellular calcium concentrations measured in 4Y5 experiments are shown. (C) Representative tracings obtained in neutrophils stimulated with
chemoattractant alone (10 nM fMLP or 10 nM IL-8) or with nucleotide (10 mM UTP or 10 mM ATP) followed by chemoattractant. The reagents were
added to cell suspensions where indicated on each individual tracing.
Nucleotides potentiate the increase of free cytosolic calcium triggered by fMLP or IL-8 in a dose-dependent manner. (A) and (B) Human
The priming effect of nucleotides in human neutrophils
363